The central concept underlying this proposal is that a microtubule based motility pathway called intraflagellar transport (IFT) is critical for the assembly and maintenance of photoreceptor outer segments (OSs). IFT is a bi-directional pathway that uses the doublet microtubules of ciliary axonemes as tracks. IFT has three essential components;a large, multiprotein complex called the IFT particle, at least one plus end directed motor (ie, kinesin II), and at least one minus end directed motor (i.e., dynein 2). During the previous grant period we demonstrated the presence of all three components in vertebrate photoreceptors and established a critical role for one component (IFT88) in OS assembly. These findings suggested that IFT plays a specific role in trafficking key components of the photo-transduction system. This competing renewal is directed at determining which photo-transduction components are transported by IFT. Preliminary data showing co-isolation of the photoreceptor membrane guanylyl cyclase 1 (GC1) and rhodopsin with IFT proteins suggests the existence of IFT-cargo complexes. This proposal is directed at detailed analysis of IFT-cargo complexes than can be isolated by immunoprecipitation. In Specific Aim 1 we will isolate hypothetical IFT-cargo complexes and determine their global composition by proteomic analysis. In Specific Aim 2 we will test the specific hypothesis that newly synthesized OS membrane proteins (ie., GC-1, rhodopsin) associate specifically with IFT-cargo complexes using a pulse-chase paradigm with 35S-methionine. In Specific Aim 3 we will test the hypothesis that the Dnaj chaperone, MRJ, mediates interaction of GC1 with the IFT particle using shRNA to knock-down the expression of MRJ and over expression of mutated and truncated forms of IFT88 and GC1. This will be done in stably transfected IMCD3 cells expressing photoreceptor GC1-GFP in the sensory cilium. Completion of these goals will provide molecular insight into the essential role for IFT in assembly of the photoreceptor outer segment. PUBLIC HEATH RELEVANCE: The photoreceptor outer segment contains hundreds of proteins that are essential for detection of light in vision. The mechanism by which the cell targets proteins necessary for vision to the outer segment are not known, but failed targeting can result in degeneration of photoreceptors and blindness. The goal of this research project is to determine the mechanism of targeting of a protein (membrane guanylyl cyclase) that when disrupted genetically leads to severe photoreceptor degeneration and blindness.